Identification Of Target Proteins Research Articles

Development of Mitochondria‐Targeting Photosensitizers via Topoisomerase I Inhibition

AbstractPhotodynamic therapy (PDT) is an important oncologic intervention option for combating drug resistance of current chemotherapy in cancer. To improve spatiotemporal control, the development of subcellular organelle‐targeting photosensitizers is critical due to the diffusion limits of short‐lived ROS. Mitochondrial topoisomerase 1 (TOP1MT), exclusively localized in mitochondria, is emerging as a promising target for anticancer treatment. In this work, we designed and synthesized mitochondria‐targeting photosensitizers via Top1 inhibition activity of IQ6 and visualized the covalent adducts between the engaged proteins and the synthesized photoaffinity labeling probes (IQ‐Ps). Positively charged derivatives (IQ‐Ss) were also synthesized and their photo‐induced cytotoxicity was assessed to select IQ‐S1. Based on the cellular accumulation of IQ‐S1 in mitochondria and the enhanced PDT activity, we demonstrated that IQ‐S1 functions as a photosensitizer with sequential targeting of mitochondria and nucleus depending on irradiation times. Our findings suggest that the translocation of IQ‐S1 is related to the damage to mitochondrial Top1. The synthesized IQ‐S1 may contribute to the identification and tracking of cellular target proteins along with the PDT progression.

Identification of Potential Protein Targets in Extracellular Vesicles Isolated from Chemotherapy-Treated Ovarian Cancer Cells.

Despite the ongoing clinical trials and the introduction of novel treatments over the past few decades, ovarian cancer remains one of the most fatal malignancies in women worldwide. Platinum- and paclitaxel-based chemotherapy is effective in treating the majority of patients with ovarian cancer. However, more than 70% of patients experience recurrence and eventually develop chemoresistance. To improve clinical outcomes in patients with ovarian cancer, novel technologies must be developed for identifying molecular alterations following drug-based treatment of ovarian cancer. Recently, extracellular vesicles (EVs) have gained prominence as the mediators of tumor progression. In this study, we used mass spectrometry to identify the changes in EV protein signatures due to different chemotherapeutic agents used for treating ovarian cancer. By examining these alterations, we identified the specific protein induction patterns of cisplatin alone, paclitaxel alone, and a combination of cisplatin and paclitaxel. Specifically, we found that drug sensitivity was correlated with the expression levels of ANXA5, CD81, and RAB5C in patients receiving cisplatin with paclitaxel. Our findings suggest that chemotherapy-induced changes in EV protein signatures are crucial for the progression of ovarian cancer.

Molecular scaffold recognition of drug molecules against essential genes of Leishmania donovani using biocomputing approach

Leishmania donovani, an obligatory intracellular flagellate pathogen, is the underlying cause of visceral leishmaniasis (VL), a fatal disease that poses a significant challenge to existing therapeutic approaches and leads to human mortality. In an endeavor to find an antileishmanial drug to combat VL, we aimed to assess the approved drug molecules against the specific drug targets of VL. In this study, a theoretical method was used to select two essential therapeutic targets (pyridoxal kinase [PK] and sterol alpha-14 demethylase [SDM]) which were present in both the data set of essential genes and drug target proteins. The selected PK and SDM proteins in L. donovani play pivotal roles as essential enzymes in the crucial vitamin B6 salvage and sterol biosynthesis pathways, respectively, leading to pathogenicity in humans. In addition to that drugs were gathered from the DrugBank and Drug Central databases and 325 (out of 4867) compounds having anti-parasitic properties were screened by PASS analysis. Consequently, three ligands (referred to as Lig_1, Lig_2, and Lig_3) were chosen based on their elevated Pa values, docking scores, and notable medicinal applications. Moreover, the result obtained from MD simulation suggests Lig_1 [Nitazoxanide (PubChem ID-41684)] does not affect the structural integrity of both targets. Additionally, evaluation of total binding energies by MMPBSA analysis showed stronger binding of Lig_1 with PK and SDM is -100.71 and -175.61 kJ/mol, respectively compared to others. As a whole, the methodology employed in this research involves the simultaneous identification of suitable protein targets and potential inhibitors. Through this investigation, we have demonstrated that compounds derived from a biocomputing approach exhibit interaction mechanisms as inhibitors against drug targets, offering a promising avenue for addressing VL.

A computational approach to fighting type 1 diabetes by targeting 2C Coxsackie B virus protein with flavonoids.

Autoimmune diabetes, well-known as type 1 insulin-dependent diabetic mellitus (T1D). T1D is a prolonged condition marked by an inadequate supply of insulin. The lack is brought on by pancreatic cell death and results in hyperglycemia. The immune system, genetic predisposition, and environmental variables are just a few of the many elements that contribute significantly to the pathogenicity of T1D disease. In this study, we test flavonoids against Coxsackie virus protein to cope the type 1 diabetes. After protein target identification we perform molecular docking of flavonoids and selected target (1z8r). then performed the ADMET analysis and select the top compound the base of the docking score and the ADMET test analysis. Following that molecular dynamics simulation was performed up to 300 ns. Root means square deviation, root mean square fluctuation, secondary structure elements, and protein-ligand contacts were calculated as post-analysis of simulation. We further check the binding of the ligand with protein by performing MM-GBSA every 10 ns. Lead compound CID_5280445 was chosen as a possible medication based on analysis. The substance is non-toxic, meets the ADMET and BBB likeness requirements, and has the best interaction energy. This work will assist researchers in developing medicine and testing it as a treatment for Diabetes Mellitus Type 1 brought on by Coxsackie B4 viruses by giving them an understanding of chemicals against these viruses.

A Graphene-Coated Silicon Wafer Plate Improves the Sensitivity and Reproducibility of MALDI-TOF MS Analysis of Proteins.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is widely used to analyze small and large molecules. However, proteins are difficult to analyze with MALDI-TOF MS in clinical applications because of their low ionization efficiency and heterogeneous crystallization with the matrix on the sample spots. Here, we investigate the potential of a customized graphene-coated silicon wafer (G/SiO2) plate for MALDI-TOF MS analysis of a clinically important protein, KPC-2, in comparison with a conventional stainless steel (SUS) plate. Our results demonstrate that the G/SiO2 plate outperforms the SUS plate in terms of sensitivity, reproducibility, and mass accuracy/precision across a wide range of molecular weights, even with highly complex samples. Furthermore, a five-day robustness test confirms the practical applicability of the G/SiO2 plate for the reliable identification of target protein(s) in MALDI-TOF MS analysis. Overall, our findings suggest that the use of the G/SiO2 plate holds great potential for improving the sensitivity and reproducibility of MALDI-TOF MS analysis for the identification of proteins, making it a promising tool for clinical applications.

The identification of two M20B family peptidases required for full virulence in Staphylococcus aureus.

We have previously demonstrated that deletion of an intracellular leucine aminopeptidase results in attenuated virulence of S. aureus. Herein we explore the role of 10 other aminopeptidases in S. aureus pathogenesis. Using a human blood survival assay we identified mutations in two enzymes from the M20B family (PepT1 and PepT2) as having markedly decreased survival compared to the parent. We further reveal that pepT1, pepT2 and pepT1/2 mutant strains are impaired in their ability to resist phagocytosis by, and engender survival within, human macrophages. Using a co-infection model of murine sepsis, we demonstrate impairment of dissemination and survival for both single mutants that is even more pronounced in the double mutant. We show that these enzymes are localized to the cytosol and membrane but are not necessary for peptide-based nutrition, a hallmark of cell-associated aminopeptidases. Furthermore, none of the survival defects appear to be the result of altered virulence factor production. An exploration of their regulation reveals that both are controlled by known regulators of the S. aureus virulence process, including Agr, Rot and/or SarA, and that this cascade may be mediated by FarR. Structural modeling of PepT1 reveals it bears all the hallmarks of a tripeptidase, whilst PepT2 differs significantly in its catalytic pocket, suggesting a broader substrate preference. In sum, we have identified two M20B aminopeptidases that are integral to S. aureus pathogenesis. The future identification of protein and/or peptide targets for these proteases will be critical to understanding their important virulence impacting functions.

Thiomethyltetrazines Are Reversible Covalent Cysteine Warheads Whose Dynamic Behavior can be "Switched Off" via Bioorthogonal Chemistry Inside Live Cells.

Electrophilic small molecules that can reversibly modify proteins are of growing interest in drug discovery. However, the ability to study reversible covalent probes in live cells can be limited by their reversible reactivity after cell lysis and in proteomic workflows, leading to scrambling and signal loss. We describe how thiomethyltetrazines function as reversible covalent warheads for cysteine modification, and this dynamic labeling behavior can be "switched off" via bioorthogonal chemistry inside live cells. Simultaneously, the tetrazine serves as a bioorthogonal reporter enabling the introduction of tags for fluorescent imaging or affinity purification. Thiomethyltetrazines can label isolated proteins, proteins in cellular lysates, and proteins in live cells with second-order rate constants spanning 2 orders of magnitude (k2, 1-100 M-1 s-1). Reversible modification by thiomethyltetrazines can be switched off upon the addition of trans-cyclooctene in live cells, converting the dynamic thiomethyltetrazine tag into a Diels-Alder adduct which is stable to lysis and proteomic workflows. Time-course quenching experiments were used to demonstrate temporal control over electrophilic modification. Moreover, it is shown that "locking in" the tag through Diels-Alder chemistry enables the identification of protein targets that are otherwise lost during sample processing. Three probes were further evaluated to identify unique pathways in a live-cell proteomic study. We anticipate that discovery efforts will be enabled by the trifold function of thiomethyltetrazines as electrophilic warheads, bioorthogonal reporters, and switches for "locking in" stability.

Transcriptomic analysis reveals novel age-independent immunomodulatory proteins as a mode of cerebroprotection in P2X4 receptor knockout mice after ischemic stroke.

Identification of new potential drug target proteins and their plausible mechanisms for stroke treatment is critically needed. We previously showed that genetic deletion and short-term pharmacological inhibition of P2X4, a purinergic receptor for adenosine triphosphate (ATP), provides acute cerebroprotection. However, potential mechanisms remain unknown. Therefore, we employed RNA-Seq technology to identify the gene expression profiles and pathway analysis followed by qPCR validation of differentially expressed genes (DEGs). This analysis identified roles of DEGs in certain biological processes responsible for P2X4R-dependent cerebroprotection after stroke. We subjected both young and aged male and female global P2X4 receptor knock out (P2X4RKO) and littermate WT (WT) mice to ischemic stroke. After three days, mice were sacrificed, and total RNA was isolated using Trizol and subjected to RNA-Seq and NanoString-mediated qPCR. DESeq2, Gene Ontology (GO), and Ingenuity Pathway Analysis (IPA) were used to identify gene expression profiles and biological pathways. We found 2246 DEGs in P2X4R KO vs. WT tissue after stroke. Out of these DEGs, 1920 genes were downregulated and 325 genes were upregulated in P2X4R KO. GO/IPA analysis of the top 300 DEGs suggests an enrichment of inflammation and extracellular matrix component genes. qPCR validation of the top 30 DEGs revealed downregulation of two common age-independent genes in P2X4R KO mice: Interleukin-6 (Il-6), an inflammatory cytokine, and Cytotoxic T Lymphocyte-Associated Protein 2 alpha (Ctla2a), an immunosuppressive factor. These data suggest that P2X4R-mediated cerebroprotection after stroke is initiated by attenuation of immune modulatory pathways in both young and aged mice of both sexes.

Mapping Peptide-Protein Interactions by Amine-Reactive Cleavable Photoaffinity Reagents.

Photoaffinity labeling followed by tandem mass spectrometry is an often used strategy to identify protein targets of small-molecule drugs or drug candidates, which, under ideal conditions, enables the identification of the actual drug binding site. In the case of bioactive peptides, however, identifying the distinct binding site is hampered because of complex fragmentation patterns during tandem mass spectrometry. We here report the development and use of small cleavable photoaffinity reagents that allow functionalization of bioactive peptides for light-induced covalent binding to their protein targets. Upon cleavage of the covalently linked peptide drug, a chemical remnant of a defined mass remains on the bound amino acid, which is then used to unambiguously identify the drug binding site. Applying our approach to known peptide-drug/protein pairs with reported crystal structures, such as the calmodulin-melittin interaction, we were able to validate the identified binding sites based on structural models. Overall, our cleavable photoaffinity labeling strategy represents a powerful tool to enable the identification of protein targets and specific binding sites of a wide variety of bioactive peptides in the future.

COVID-19 progression towards ARDS: a genome wide study reveals host factors underlying critical COVID-19.

Coronavirus disease 2019 (COVID-19) is a viral infection produced by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus epidemic, which was declared a global pandemic in March 2020. The World Health Organization has recorded around 43.3 billion cases and 59.4 million casualties to date, posing a severe threat to global health. Severe COVID-19 indicates viral pneumonia caused by the SARS-CoV-2 infections, which can induce fatal consequences, including acute respiratory distress syndrome (ARDS). The purpose of this research is to better understand the COVID-19 and ARDS pathways, as well as to find targeted single nucleotide polymorphism. To accomplish this, we retrieved over 100 patients' samples from the Sequence Read Archive, National Center for Biotechnology Information. These sequences were processed through the Galaxy server next generation sequencing pipeline for variant analysis and then visualized in the Integrative Genomics Viewer, and performed statistical analysis using t-tests and Bonferroni correction, where six major genes were identified as DNAH7, CLUAP1, PPA2, PAPSS1, TLR4, and IFITM3. Furthermore, a complete understanding of the genomes of COVID-19-related ARDS will aid in the early identification and treatment of target proteins. Finally, the discovery of novel therapeutics based on discovered proteins can assist to slow the progression of ARDS and lower fatality rates.

The Introduction of Detergents in Thermal Proteome Profiling Requires Lowering the Applied Temperatures for Efficient Target Protein Identification.

Although the use of detergents in thermal proteome profiling (TPP) has become a common practice to identify membrane protein targets in complex biological samples, surprisingly, there is no proteome-wide investigation into the impacts of detergent introduction on the target identification performance of TPP. In this study, we assessed the target identification performance of TPP in the presence of a commonly used non-ionic detergent or a zwitterionic detergent using a pan-kinase inhibitor staurosporine, our results showed that the addition of either of these detergents significantly impaired the identification performance of TPP at the optimal temperature for soluble target protein identification. Further investigation showed that detergents destabilized the proteome and increased protein precipitation. By lowering the applied temperature point, the target identification performance of TPP with detergents is significantly improved and is comparable to that in the absence of detergents. Our findings provide valuable insight into how to select the appropriate temperature range when detergents are used in TPP. In addition, our results also suggest that the combination of detergent and heat may serve as a novel precipitation-inducing force that can be applied for target protein identification.

Use of automated capillary immunoassays for quantification of antibodies in chicken sera against recombinant Salmonella enterica serotype Heidelberg proteins

The classic immunoblot technique is an important tool for identification and characterization of target proteins. However, a standard protocol for this classic immunoblot assay involves many steps that may cause experimental variations in each step and make quantification of antibodies in sera difficult. A capillary electrophoresis-based immunoblot system was developed to reduce potential problems in variations during the experimental process, enable protein identification in an automatic manner and quantitate various isotypes of antibodies in sera. In the present study, we used this system to examine the purity of the recombinant proteins and measure amounts of various isotypes of immunoglobins in chicken sera after immunization with two recombinant Salmonella FliD and FimA proteins. A single band of each protein was detected in the gel like images by this system after purification by nickel-chelated affinity chromatography. A good linear range of the protein concentrations was also obtained for each recombinant protein. This automated capillary immunoblot system was successfully used for detection and quantification of various immunoglobin isotypes against two recombinant Salmonella proteins from the immunized chicken sera, but not the un-immunized chicken sera. The chicken immunoglobin G (IgG) antibody response to the FliD protein from the immunized group was 1110- and 51,400-fold higher than that from the un-immunized chickens both two- and three-weeks post-vaccination, respectively. It was also observed that IgM antibody against the FliD protein from the immunized chickens was 1030-fold higher than that from the un-immunized chickens two weeks post-vaccination, but the IgM response declined to 120-fold between two groups from two weeks to three weeks after immunization. The IgM antibody response to the FimA protein from the immunized group was 1.84- and 1.12-fold higher than that from the un-immunized group, respectively, both two- and three-weeks post-vaccination, while the IgG antibody response from the immunized group was 8.07- and 27.6-fold higher than that from the un-immunized group, respectively, during the same period. These results suggest that this capillary-based immunoblot assay can be an alternative method for analyses and quantitation of chicken humoral immune response before and after immunization with any antigens and/or for investigation in Salmonella outbreaks.

Isolation and identification of the new baicalin target protein to develop flavonoid structure-based therapeutic agents

Proteins are vital constituents of all living organisms. As many therapeutic agents alter the activity of functional proteins, identifying functional target proteins of small bioactive molecules isessential for the rational design of stronger medicines. Flavonoids with antioxidant, anti-allergy, and anti-inflammatory effects are expected to have preventive effects for several diseases closely related to oxidation and inflammation, including heart disease, cancer, neurodegenerative disorders, and eye diseases. Therefore, identifying the proteins involved in the pharmacological actions of flavonoids, and designing a flavonoid structure-based medicine that strongly and specifically inhibits flavonoid target proteins, could aid the development of more effective medicines for treating heart disease, cancer, neurodegenerative disorders, and ocular diseases with few side effects. To isolate the flavonoid target protein, we conducted a novel affinity chromatography in a column wherein baicalin, a representative flavonoid, was attached to Affi-Gel 102. Through affinity chromatography and nano LC-MS/MS, we identified GAPDH as a flavonoid target protein. Then, we performed fluorescence quenching and an enzyme inhibition assay to experimentally confirmbaicalin’s binding affinity for, and inhibition of, GAPDH. We also conducted in silico docking simulations to visualize the binding modes of baicalin and the newly identified flavonoid target protein, GAPDH. From the results of this study, it was considered that one of the reasons why baicalin exhibits the effects on cancer and neurodegenerative diseases is that it inhibits the activity of GAPDH. In summary, we showed that Affi-Gel102 could quickly and accurately isolate the target protein for bioactive small molecules, without the need for isotopic labeling or a fluorescent probe. By using the method presented here, it was possible to easily isolate the target protein of a medicine containing a carboxylic acid.

Identification of RNA-Binding Protein Targets with HyperTRIBE in Saccharomyces cerevisiae.

As a master regulator in cells, RNA-binding protein (RBP) plays critical roles in organismal development, metabolism and various diseases. It regulates gene expression at various levels mostly by specific recognition of target RNA. The traditional CLIP-seq method to detect transcriptome-wide RNA targets of RBP is less efficient in yeast due to the low UV transmissivity of their cell walls. Here, we established an efficient HyperTRIBE (Targets of RNA-binding proteins Identified By Editing) in yeast, by fusing an RBP to the hyper-active catalytic domain of human RNA editing enzyme ADAR2 and expressing the fusion protein in yeast cells. The target transcripts of RBP were marked with new RNA editing events and identified by high-throughput sequencing. We successfully applied HyperTRIBE to identifying the RNA targets of two yeast RBPs, KHD1 and BFR1. The antibody-free HyperTRIBE has competitive advantages including a low background, high sensitivity and reproducibility, as well as a simple library preparation procedure, providing a reliable strategy for RBP target identification in Saccharomyces cerevisiae.

CETSA and thermal proteome profiling strategies for target identification and drug discovery of natural products

BackgroundMonitoring target engagement at various stages of drug development is essential for natural product (NP)-based drug discovery and development. The cellular thermal shift assay (CETSA) developed in 2013 is a novel, broadly applicable, label-free biophysical assay based on the principle of ligand-induced thermal stabilization of target proteins, which enables direct assessment of drug-target engagement in physiologically relevant contexts, including intact cells, cell lysates and tissues. This review aims to provide an overview of the work principles of CETSA and its derivative strategies and their recent progress in protein target validation, target identification and drug lead discovery of NPs. MethodsA literature-based survey was conducted using the Web of Science and PubMed databases. The required information was reviewed and discussed to highlight the important role of CETSA-derived strategies in NP studies. ResultsAfter nearly ten years of upgrading and evolution, CETSA has been mainly developed into three formats: classic Western blotting (WB)-CETSA for target validation, thermal proteome profiling (TPP, also known as MS-CETSA) for unbiased proteome-wide target identification, and high-throughput (HT)-CETSA for drug hit discovery and lead optimization. Importantly, the application possibilities of a variety of TPP approaches for the target discovery of bioactive NPs are highlighted and discussed, including TPP-temperature range (TPP-TR), TPP-compound concentration range (TPP-CCR), two-dimensional TPP (2D-TPP), cell surface-TPP (CS-TPP), simplified TPP (STPP), thermal stability shift-based fluorescence difference in 2D gel electrophoresis (TS-FITGE) and precipitate supported TPP (PSTPP). In addition, the key advantages, limitations and future outlook of CETSA strategies for NP studies are discussed. ConclusionThe accumulation of CETSA-based data can significantly accelerate the elucidation of the mechanism of action and drug lead discovery of NPs, and provide strong evidence for NP treatment against certain diseases. The CETSA strategy will certainly bring a great return far beyond the initial investment and open up more possibilities for future NP-based drug research and development.

Retraction: Proteomics Approaches for Identification of Tumor Relevant Protein Targets in Pulmonary Squamous Cell Carcinoma by 2D-DIGE-MS.

Retraction: Proteomics Approaches for Identification of Tumor Relevant Protein Targets in Pulmonary Squamous Cell Carcinoma by 2D-DIGE-MS.

Abstract 1180: Identification of target proteins for breast cancer genetic risk loci and blood risk biomarkers in a large study by integrating genomic and proteomic data

Abstract Genome-wide association studies (GWAS) have identified around 200 loci associated with breast cancer risk. However, protein targets for these loci remain largely unknown. Identifying protein targets and biomarkers can improve the understanding of cancer biology and etiology and identify high-risk individuals for cancer prevention. In this study, we investigated genetically predicted levels of 1,142 circulating proteins with breast cancer risk in 133,384 cases and 113,789 controls of European ancestry included in the Breast Cancer Association Consortium (BCAC). We identified 22 blood protein biomarkers associated with the risk of overall breast cancer at a false discovery rate (FDR) <0.05, including nine proteins encoded by genes located at least 500kb away from previously reported risk variants for breast cancer. Analyses focusing on 124 encoding genes located at GWAS-identified breast cancer risk loci found 20 proteins associated with overall breast cancer risk and one protein associated with triple-negative breast cancer risk at FDR <0.05. Adjustment for the GWAS-identified risk variants significantly attenuated the association for 13 of these proteins, suggesting that these proteins may be the targets of these GWAS-identified risk loci. The identified proteins are involved in various biological processes, including glutathione conjugation, STAT5 signaling, and NF-kB signaling pathways. Our study identified novel protein targets and risk biomarkers for breast cancer risk. Citation Format: Guochong Jia, Yaohua Yang, Jie Ping, Shuai Xu, Lili Liu, Xingyi Guo, Ran Tao, Jirong Long, Wei Zheng. Identification of target proteins for breast cancer genetic risk loci and blood risk biomarkers in a large study by integrating genomic and proteomic data [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1180.

Identification of Novel Protein Targets of Prodigiosin for Breast Cancer Using Inverse Virtual Screening Methods.

Prodigiosin (PG) is chemically formulated as 4-methoxy-5-[(5-methyl-4-pentyl-2H-pyrrol-2ylidene)methyl]-2,2'-bi-1H-pyrrole and it is an apoptotic agent. Only a few protein targets for PG have been identified so far for regulating various diseases; nevertheless, finding more PG targets is crucial for novel drug discovery research. A bioinformatics method was applied in this work to find additional potential PG targets. Initially, a text mining analysis was conducted to determine the relationship between PG and a variety of metabolic processes. One hundred sixteen proteins from the KEGG pathway were selected for the docking study. Inverse virtual screening was performed by Discovery Studio software 4.1 using CHARMm-based docking tool. Twelve proteins are screened out of 116 because their CDOCKER interaction energy is larger than - 40.22kcal/mol. The best docking score with PG was reported to be - 44.25kcal/mol, - 44.99kcal/mol, and - 40.91kcal/mol for three novel proteins, such as human epidermal growth factor-2 (HER-2), mitogen-activated protein kinase (MEK), and S6 kinase protein (S6K) respectively. The interactions in the S6K/PG complex are predominantly hydrophobic; however, hydrogen bond interactions can be identified in the MEK/PG and HER-2/PG complexes. The root-mean-square deviation (RMSD) and key interaction score system (KISS) were further used to validate the docking approach. The docking approach employed in this work has a low RMSD value (2.44Å) and a high KISS score (0.5), indicating that it is significant.

Verification of the Interaction Target Protein of the Effector ApCE22 of Arthrinium phaeospermum in Bambusa pervariabilis × Dendrocalamopsis grandis.

The study of interaction proteins of the pathogen A. phaeospermum effector protein is an important means to analyze the disease-resistance mechanism of Bambusa pervariabilis × Dendrocalamopsis grandis shoot blight. To obtain the proteins interacting with the effector ApCE22 of A. phaeospermum, 27 proteins interacting with the effector ApCE22 were initially identified via a yeast two-hybrid assay, of which four interaction proteins were obtained after one-to-one validation. The B2 protein and the chaperone protein DnaJ chloroplast protein were then verified to interact with the ApCE22 effector protein by bimolecular fluorescence complementation and GST pull-down methods. Advanced structure prediction showed that the B2 protein contained the DCD functional domain related to plant development and cell death, and the DnaJ protein contained the DnaJ domain related to stress resistance. The results showed that both the B2 protein and DnaJ protein in B. pervariabilis × D. grandis were the target interaction proteins of the ApCE22 effector of A. phaeospermum and related to the stress resistance of the host B. pervariabilis × D. grandis. The successful identification of the pathogen effector interaction target protein in B. pervariabilis × D. grandis plays an important role in the mechanism of pathogen-host interaction, thus providing a theoretical basis for the control of B. pervariabilis × D. grandis shoot blight.

Identification of Target Proteins for Promoting Nuclear Envelope Rupture in Cancer Cells

The nuclear envelope separates and protects DNA from the cytoplasm. In cancer cells, frequent nuclear morphological abnormalities and transient rupture of the nuclear membrane promote DNA damage and cancer malignancy. However, nuclear envelope instability, acting as a tumor-promotion factor, can be viewed as a fatal weakness of cancer cells. Although the exact mechanism is not clear, it is known that the loss of the tumor suppressor gene TP53 is involved in nuclear envelope instability in cancer cells. In this study, we demonstrate that the inhibition of NUP93, which is a subunit of the nuclear pore complex, induces cell death by enhancing nuclear envelope instability. The RNA interference (RNAi) inhibiting NUP93 expression in the human cell line RPE1 caused nuclear envelope rupture, and DNA damage, leading to cell death. Under the condition in which the tumor suppressor gene TP53 was simultaneously suppressed with NUP93, nuclear envelope rupture and DNA damage were significantly increased. Conversely, cell death was slightly decreased. The decrease in cell death may be ascribed to the fact that TP53 is involved in the induction of apoptosis by DNA damage. We speculate that amplification of nuclear envelope instability may induce cancer cell-specific synthetic lethality in the context of TP53-independent nuclear envelope instability. Moreover, our results suggest that nuclear pore proteins can be promising targets for the development of synthetic lethal anticancer drugs.